Affiliation: Translational Cancer Research Group, School of Medical and Molecular Biosciences, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia. Eileen.Mcgowan@uts.edu.au

ABSTRACTEndocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

pone-0020623-g003: EdU incorporation post ICI 182780 and IPTG (p14ARF) treatment.Cells were seeded on cover slips and treated with 5 mM IPTG or 10 nM ICI 182780 24 h post-seeding. EdU was added to the medium on days 1 and 4 and cells were incubated for a further 20 h. EdU incorporation was visualized by staining with Alexafluor 488 (green). The nucleus was stained with Hoechst 33342 (blue) and images were taken on a Nikon fluorescence microscope (magnification ×200). Column graph shows % cells staining for EdU compared to Hoechst 33342 stained nuclei (± SE). Experiments were performed in triplicate (duplicate biological experiments). A minimum of 500 cells was counted for each treatment.

Mentions:
To measure DNA synthesis, in particular the percentage of cells in S-phase, EdU was added directly to the MCF-7p14ARF cell culture medium 24 h and 96 h post-treatment with IPTG or ICI 182780 and cells were incubated for a further 20 h. This allowed the visualization of individual cell proliferation captured over a 20-hour window. At 24 h post-IPTG treatment, 5% of IPTG-treated cells synthesized DNA (Fig. 3), suggesting rapid repression of DNA synthesis upon reactivation of the p14ARF pathway in MCF-7 cells, which is consistent with the cell count data, but not with MTS or SYBR green assays (Fig. 1). Reduced proliferation was maintained for 96 h–116 h (Fig. 3). These later-day results were in agreement with the cell counts and SYBR-DNA assay as shown in Figure 1, but not with the MTS assay. ICI 182780-treated cells showed a significant inhibition in DNA synthesis by day 4–5 post-treatment when only 20% of these cells incorporated EdU when compared to control cells (P<0.0001; Fig. 3). Interestingly, ICI 182780 treatment showed a delayed cell cycle arrest mechanism when compared to p14ARF induction with IPTG, which showed an early inhibition of DNA synthesis by day 1–2 (P<0.0001; Fig. 3). This is consistent with the Flow analysis data and previously reported literature where ICI 182780 effective blocks the cell cycle only in G1/G0 phase [2].

pone-0020623-g003: EdU incorporation post ICI 182780 and IPTG (p14ARF) treatment.Cells were seeded on cover slips and treated with 5 mM IPTG or 10 nM ICI 182780 24 h post-seeding. EdU was added to the medium on days 1 and 4 and cells were incubated for a further 20 h. EdU incorporation was visualized by staining with Alexafluor 488 (green). The nucleus was stained with Hoechst 33342 (blue) and images were taken on a Nikon fluorescence microscope (magnification ×200). Column graph shows % cells staining for EdU compared to Hoechst 33342 stained nuclei (± SE). Experiments were performed in triplicate (duplicate biological experiments). A minimum of 500 cells was counted for each treatment.

Mentions:
To measure DNA synthesis, in particular the percentage of cells in S-phase, EdU was added directly to the MCF-7p14ARF cell culture medium 24 h and 96 h post-treatment with IPTG or ICI 182780 and cells were incubated for a further 20 h. This allowed the visualization of individual cell proliferation captured over a 20-hour window. At 24 h post-IPTG treatment, 5% of IPTG-treated cells synthesized DNA (Fig. 3), suggesting rapid repression of DNA synthesis upon reactivation of the p14ARF pathway in MCF-7 cells, which is consistent with the cell count data, but not with MTS or SYBR green assays (Fig. 1). Reduced proliferation was maintained for 96 h–116 h (Fig. 3). These later-day results were in agreement with the cell counts and SYBR-DNA assay as shown in Figure 1, but not with the MTS assay. ICI 182780-treated cells showed a significant inhibition in DNA synthesis by day 4–5 post-treatment when only 20% of these cells incorporated EdU when compared to control cells (P<0.0001; Fig. 3). Interestingly, ICI 182780 treatment showed a delayed cell cycle arrest mechanism when compared to p14ARF induction with IPTG, which showed an early inhibition of DNA synthesis by day 1–2 (P<0.0001; Fig. 3). This is consistent with the Flow analysis data and previously reported literature where ICI 182780 effective blocks the cell cycle only in G1/G0 phase [2].

Bottom Line:
Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation.We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells.These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

Affiliation:
Translational Cancer Research Group, School of Medical and Molecular Biosciences, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia. Eileen.Mcgowan@uts.edu.au

ABSTRACTEndocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.